Nanopore device and method of manufacturing same

ABSTRACT

A 3D nanopore device for characterizing biopolymer molecules includes a first selecting layer having a first axis of selection. The device also includes a second selecting layer disposed adjacent the first selecting layer and having a second axis of selection orthogonal to the first axis of selection. The device further includes an third electrode layer disposed adjacent the second selecting layer, such that the first selecting layer, the second selecting layer, and the third electrode layer form a stack of layers along a Z axis and define a plurality of nanopore pillars.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 16/147,362, filed on Sep. 28, 2018 under attorney docket no.BTL.20001.00, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/566,313, filed on Sep. 29, 2017 under attorneydocket number 165-101USIP and entitled “MANUFACTURE OF THREE DIMENSIONALNANOPORE DEVICE,” and U.S. Provisional Patent Application Ser. No.62/593,840, filed on Dec. 1, 2017 under attorney docket numberBTL.30002.00 and entitled “NANOPORE DEVICE AND METHOD OF MANUFACTURINGSAME.” This application includes subject matter similar to the subjectmatter described in co-owned U.S. Provisional Patent Application Ser.No. 62/612,534, filed on Dec. 31, 2017 under attorney docket numberBTL.30003.00 and entitled “NANOPORE DEVICE AND METHODS OF ELECTRICALARRAY ADDRESSING AND SENSING,” U.S. Provisional Patent Application Ser.No. 62/628,214, filed on Feb. 8, 2018 under attorney docket numberBTL.30004.00 and entitled “BIOMEMORY FOR NANOPORE DEVICE AND METHODS OFMANUFACTURING SAME,” and U.S. Provisional Patent Application Ser. No.62/711,234, filed on Jul. 27, 2018 under attorney docket numberBTL.30005.00 and entitled “NANOPORE DEVICE AND METHODS OF DETECTINGCHARGED PARTICLES USING SAME.” The contents of the applicationsmentioned herein are fully incorporated herein by reference as thoughset forth in full.

FIELD OF THE INVENTION

The present invention relates generally to systems, devices, andprocesses for characterizing biopolymer molecules, and methods ofmanufacturing such systems and devices.

BACKGROUND

Nucleic acid (e.g., DNA, RNA, etc.) sequencing is one of the mostpowerful methods to identify genetic variations at the molecular level.Many signatures of genetic diseases can be diagnosed by informationcollected through genome-wide single nucleotide polymorphisms (“SNPs”)analysis, gene fusion, genomic insertion and deletion, etc. Thesetechniques and other molecular biology techniques require nucleic acidsequencing at some point. Current technologies to sequence nucleic acidsat the single molecule level include a nanopore sequencing technologythat has advantages over previous sequencing techniques because nanoporesequencing technology has the characteristics of a label-free andamplification-free technique that also has improved read lengths, andimproved system throughput. Accordingly, nanopore sequencing technologyhas been incorporated into high-quality gene sequencing applications.

Early experimental systems for nanopore based DNA sequencing detectedelectrical behavior of ssDNA passing through an α-hemolysin (αHL)protein nanopore. Since then, nanopore based nucleic acid sequencingtechnology has been improved. For instance, solid-state nanopore basednucleic acid sequencing replaces biological/protein based nanopores withsolid state (e.g., semiconductor, metallic gates) nanopores, asdescribed below.

A nanopore is a small hole (e.g., with a diameter of about 1 nm to about1000 nm) that can detect the flow of electrons through the hole by thechange in the ionic current and/or tunneling current. Because eachnucleotide of a nucleic acid (e.g., adenine, cytosine, guanine, thyminein DNA, uracil in RNA) affects the electric current density across thenanopore in a specific manner as it physically passes through thenanopore, measuring changes in the current flowing through a nanoporeduring translocation results in data that can be used to directlysequence a nucleic acid molecule passing through the nanopore. As such,Nanopore technology is based on electrical sensing, which is capable ofdetecting nucleic acid molecules in concentrations and volumes muchsmaller than that required for other conventional sequencing methods.Advantages of nanopore based nucleic acid sequencing include long readlength, plug and play capability, and scalability. However, currentbiological nanopore based nucleic acid sequencing techniques can requirea fixed nanopore opening (e.g., with a diameter of about 2 nm), havepoor sensitivity (i.e., unacceptable amount of false negatives), highcost that renders production worthy manufacturing a challenge, andstrong temperature and concentration (e.g., pH) dependency.

With advancements in semiconductor manufacturing technologies,solid-state nanopores have become an inexpensive and superioralternative to biological nanopores partly due to the superiormechanical, chemical and thermal characteristics, and compatibility withsemiconductor technology allowing the integration with other sensingcircuitry and nanodevices. However, current nanopore DNA sequencingtechniques (e.g., involving biological and/or solid-state nanopores)continue to suffer from various limitations, including low sensitivityand high manufacturing cost. FIG. 1 schematically depicts a state-of-artsolid-state based 2-dimensional (“2D”) nanopore sequencing device 100.While, the device 100 is referred to as “two dimensional,” the device100 has some thickness along the Z axis.

Many of the limitations of nanopore DNA sequencing techniques resultfrom the intrinsic nature of nanopore devices and techniques that mustovercome the fast translocation speed and small size (e.g., height ofabout 0.34 nm and diameter of about 1 nm) of a single nucleotide.Conventional electronic instrumentation (e.g., nano-electrodes) cannotresolve such fast moving and small nucleotides using conventionalnanopore based DNA sequencing techniques. Also high manufacturing costprevents wider applications of nanopore based DNA sequencing.

Many efforts have been made to overcome these drawbacks, including theuse of many different types of biological, solid-state and hybrid(biological and solid-state) nanopores and nanopore sensors. However,none of these efforts has been successful in mass production.

There is a need for nanopore based sequencing systems and devices thataddress the shortcomings of currently-available configurations. Inparticular, there is a need for nanopore based sequencing systems anddevices with acceptable sensitivity and manufacturing cost.

SUMMARY

Embodiments described herein are directed to nanopore based sequencingsystems and methods of manufacturing same. In particular, theembodiments are directed to 3D nanopore based sequencing systems andmethods of manufacturing same.

In one embodiment, a 3D nanopore device for characterizing biopolymermolecules includes a first selecting layer having a first axis ofselection. The device also includes a second selecting layer disposedadjacent the first selecting layer and having a second axis of selectionorthogonal to the first axis of selection. The device further includesan third electrode layer disposed adjacent the second selecting layer,such that the first selecting layer, the second selecting layer, and thethird electrode layer form a stack of layers along a Z axis and define aplurality of nanopore pillars.

In one or more embodiments, the first selecting layer includes a firstplurality of inhibitory electrodes. The second selecting layer mayinclude a second plurality of inhibitory electrodes. The first andsecond pluralities of inhibitory electrodes may form an array partiallydefining the plurality of nanopore pillars therein. The third electrodelayer may include an electrode configured to modulate an electrical biasand to detect a current modulation. The device of may also include oneor more electrode layers disposed adjacent the third electrode layer.

In one or more embodiments, the device also includes a top chamberdisposed adjacent the first selecting layer. The device further includesa bottom chamber disposed adjacent a bottom electrode layer, such thatthe plurality of nanopore pillars fluidly couple the top and bottomchambers and provide a translocation channel when multi-electrodes arepresent in the device. The device may also include an electrolytesolution in the top and bottom chambers and surrounding the firstselecting layer, the second selecting layer, and the third electrodelayer. The electrolyte solution may include KCl or LiCl₂.

In one or more embodiments, the third electrode layer includes a metalrate-control electrode. The third electrode layer may include metals,such as Ta, Al, Cr, Au—Cr, Ti, Graphene, or Al—Cu. The third electrodemay include highly doped (either n+ or p+ type) polysilicon or salicidedpolysilicon. The third electrode layer may have a thickness from 0.2 nmto 1000 nm. The third electrode layer may include a sensing electrode.The sensing electrode may operate by ion blockade, tunneling, capacitivesensing, piezoelectric, or microwave-sensing.

In one or more embodiments, the device also includes an inner membranelayer configured to modify an inner diameter of the plurality ofnanopores. The inner membrane layer may include low stress silicon richnitrides, such as Si₃N₄ and is coated with dielectrics, such as Al₂O₃,SiO₂, ZnO, or HfO₂. The inner membrane layer may have a thickness ofabout 10 nm to about 50 nm. Each of the plurality of nanopores may haverespective diameters from about 0.2 nm to about 1000 nm. The device mayalso include a top membrane layer. The top membrane layer may includeSi₃N₄, Al₂O₃, SiO₂, 2D dielectrics (e.g., MoS₂ or hBN), and polymermembranes (e.g., polyimide and PDMS). The top membrane layer may have athickness of about 5 nm to about 50 nm.

In another embodiment, a method of manufacturing a 3D nanopore deviceincludes depositing a first Si₃N₄ layer on the first Si substrate or thefirst dielectric base layer. The method includes depositing a firstdielectric layer on the first Si₃N₄ layer. The method also includesdepositing a first metal or polysilicon layer on the first dielectriclayer. In one or more embodiments, the method also includes etching andpatterning the first metal or polysilicon electrode layer. The methodalso includes depositing a second dielectric layer on the patternedfirst metal or polysilicon electrode layer.

The method also includes depositing a second metal or polysilicon layeron the first metal or polysilicon electrode layer. The method furtherincludes depositing a second Si₃N₄ layer on a second dielectric layer.In one or more embodiments, the method also includes etching andpatterning the second metal or polysilicon electrode layer. The methodfurther includes depositing and patterning the multiple layers of metalor polysilicon electrode layers.

The method includes etching the first Si or dielectric substrate baselayer from the backside to create a channel from the backside.

The method includes a patterning the nanopore channel from the surfaceon the multiple stack of Si₃N₄ layers, dielectric layer, and metal orpolysilicon layers to form a nanopore therethrough. The method may alsoinclude disposing each metal or polysilicon electrodes in every channeland electrically coupling the metal or polysilicon electrode layers.

In one or more embodiments, the method also includes etching a secondchannel into the bottom Si₃N₄ layer, where the first and second channelsare orthogonal to each other. The method may also include disposing asecond inhibitory electrode in the second channel and electricallycoupling the second inhibitory electrode to the bottom Si₃N₄ layer. Themethod may also include depositing a third dielectric base layer on thesecond metal layer, and etching the third dielectric base layer to formthe nanopore therethrough. The method may also include etching the thirddielectric layer, and electrically coupling a third electrode to thethird dielectric layer. The method may also include etching a substrate,and fluidly coupling a bottom chamber to the plurality of nanoporepillars.

In one or more embodiments, the method also includes disposing the firstdielectric base layer, the first Si₃N₄ layer, and the first metal in amiddle chamber between top and bottom chambers, where the top, middle,and bottom chambers contain an electrolyte solution, such that the topand bottom chambers are fluidly coupled by the nanopore. Deposition ofthe first Si₃N₄ layer, the first metal layer, and the dielectric coatinglayer may use ALD or CVD. Etching the first dielectric base layer, thefirst Si₃N₄ layer, and the first metal layer to form the nanopore mayuse high aspect ratio etching.

In still another embodiment, a method of detecting a charged particleuses a 3D nanopore device having top, middle and bottom chambers, and a3D nanopore array disposed in the middle chambers such that the top andbottom chambers are fluidly coupled by a plurality of nanopores in the3D nanopore array. The method includes adding electrolyte solutionincluding the charged particle to the top, middle, and bottom chambers.The method also includes placing top and bottom electrodes in the topand bottom chambers respectively. The method further includes applyingan electrophoretic bias between the top and bottom electrodes. Moreover,the method includes applying first and second selection biases to firstand second selection electrodes in the 3D nanopore device to select oneor more nanopores of the plurality of nanopores through which thecharged particle will be directed. In addition, the method includesapplying a rate control bias to a rate control electrode in the 3Dnanopore device to modulate a translocation rate of the charged particlethrough the one or more nanopores. The method also includes applying asensing bias to a sensing electrode in the 3D nanopore device. Themethod further includes detecting a change in a current in the sensingelectrode.

In one or more embodiments, the current is an electrode current or atunneling current.

In yet another embodiment, a method of manufacturing a sensor includinga 3D nanopore channel pillar array, a plurality of electrodes, a topchamber, and a bottom chamber, includes placing the 3D nanopore channelpillar array in an electrolyte solution including biomolecules and DNA.The method also includes placing an electrode in the electrolyte. Themethod further includes applying a bias to the electrode in theelectrolyte. Moreover, the method includes placing cross patternedcolumn and row inhibitory electrodes surrounding nanopore pillars on topof the 3D nanopore channel pillar array. In addition, the methodincludes placing metal plane electrodes surrounding the nanopore pillarsin the 3D nanopore channel pillar array, the metal plane electrodesincluding a rate-control electrode and a sensing electrode. The methodalso includes applying a rate-control bias in the rate-controlelectrode. The method further includes applying a sensing bias in thesensing electrode. Moreover, the method includes detecting a change inan electrode current in the electrolyte. In addition, the methodincludes detecting a change in a tunneling current in the electrodes.

In one or more embodiments, the rate-control electrode has a thicknessranges from about 2 nm to about 1000 nm. The rate-control electrode mayinclude Ta, Cr, Al, Au—Cr, Graphene, or Al—Cu. It may include heavilydoped (n- or p-type) polysilicon or salicided polysilicon. The 3Dnanopore channel pillar array may include a biological layer having therate-control electrode, such that the 3D nanopore channel pillar arrayis a hybrid. The top and bottom chambers may contain at least some ofthe electrolyte solution. The electrolyte solution may include KCl andLiCl₂. The electrodes for top and bottom chamber may include Ag/AgCl₂.The cross patterned column and row inhibitory electrodes may enablearray operation by selecting and deselecting the column and row byapplying an inhibitory bias to stop ionic current flow vertically. Thesensing electrode may utilize ion blockade sensing, tunneling sensing,capacitive sensing, piezoelectric sensing, and/or wave-sensing.

In one or more embodiments, the 3D nanopore channel pillar arrayincludes a plurality of dielectric-electrodes in a dielectric-electrodestack. The dielectric-electrode stack includes a membrane layer, adielectric layer to modify a nanopore channel opening width, an array ofnanopore channel pillars, a stack of rate-control dielectric-electrodelayers, a stack of sensing dielectric-electrode layers, and a sourceselect dielectric-electrode layer. The membrane layer may include adielectric material and have a thickness from about 10 nm to about 50nm. The dielectric material may be Si₃N₄, Al₂O₃, or SiO₂. The membranelayer may modify the nanopore channel opening width. The nanoporechannel opening width may be from about 2 nm to about 100 nm patternedby standard optical lithography and ion beam (e.g., FIB, TEM)techniques.

In one or more embodiments, the 3D nanopore channel pillar array ismanufactured using ALD and/or CVD deposition of dielectric layers, Highaspect ratio Reactive Ion Etch deep trench process (nanopore channelopening etch), ALD and/or CVD deposition of trimming dielectric layers,and/or ALD and/or CVD deposition of membrane dielectric layers.

In one or more embodiments, the dielectric-electrode stack also includesa bottom dielectric layer. The bottom dielectric layer may have athickness of about 100 nm to 1000 nm. The bottom dielectric layer mayinclude SiO₂, glass, or SOI to reduce substrate coupled low level noise.The dielectric-electrode stack may also include a top dielectric layer.The top dielectric layer may have a thickness of about 5 nm to about 50nm. The top dielectric layer may include SiO₂, Si₃N₄, or Al₂O₃. The topdielectric layer may determine a final nanopore channel opening width.

In one or more embodiments, the method also includes forming a nanoporechannel pillar using high aspect ratio etching to provide a sharp shapeto a trench profile of the nanopore channel pillar. The high aspectratio etching may have an aspect ratio of greater than 5.

In one or more embodiments, the 3D nanopore channel pillar arrayfacilitates multiplex sequencing applications using high density lowcost nanopore channels. A number of electrodes in the 3D nanopore arraymay be selected depending on the required sequencing applications toprovide a Time of Flight (“TOF”) technique to control a translocationspeed by controlled biasing. Controlling the translocation speed mayimprove reading of DNA molecules and improves a sensitivity of thesensor.

In one or more embodiments, the 3D nanopore channel pillar array isintegrated in a CMOS flow, thereby facilitating embedded biosensorsolutions for CMOS technology. The CMOS flow may include a 2-dim wellfor electrochemical reactions. The CMOS flow may include anion-sensitive filed effect transistor technology.

In one or more embodiments, the 3D nanopore channel pillar arrayincorporates a hybrid type of nanopore technology including a biologicalcomponent and a solid-state component in a 3D configuration. The 3Dnanopore channel pillar array may facilitate an electro-chemical,thermal, or electro-optical reaction to take place in an enlarged,separated nanopore well with a multi-electrode system to enhanceelectrochemical and/or sequencing reactions. The 3D nanopore channelpillar array may facilitate multiplex sequencing using a multi-arrayconfiguration where individual nanopore channel pillars are addressable.The 3D nanopore channel pillar array may facilitate standard qPCR withina nanopore channel pillar. The 3D nanopore channel pillar array mayfacilitate probe-mediated targeted sequencing. The 3D nanopore channelpillar array may facilitate tuning of a nanopore channel opening widthfor different applications.

In one or more embodiments, the nanopore channel opening width istunable from about 1 nm to about 100 nm. The nanopore channel openingwidth may be electronically tunable during manufacturing.

In one or more embodiments, the method also includes forming a hybridnanopore channel to enhance stability of the sensor. Forming the hybridnanopore may include inserting a stable biological component toconstruct a semi-synthetic membrane porin. The stable biologicalcomponent may be an αHL molecule. The αHL molecule may be inserted intoa SiN based 3D nanopore.

In one or more embodiments, the method also includes using a topinhibitory electrode to induce a structure in the stable biologicalcomponent to ensure alignment of the stable biological component andhybrid nanopore.

The aforementioned and other embodiments of the invention are describedin the Detailed Description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects of embodiments are described in furtherdetail with reference to the accompanying drawings, in which the sameelements in different figures are referred to by common referencenumerals, wherein:

FIG. 1 schematically illustrates a prior art solid-state 2D nanoporedevice;

FIGS. 2A-2D schematically illustrate a 3D nanopore device according toone embodiment from perspective, top, front, and right views,respectively.

FIG. 3 schematically illustrates a 3D nanopore device according to oneembodiment including some details of its operation.

FIG. 4 is a table summarizing the voltage operation of the nanoporedevice depicted in FIG. 3.

FIG. 5 schematically illustrates a 3D nanopore device according to oneembodiment including some of the electrodes therein.

FIGS. 6A-6E illustrate a method for manufacturing a 3D nanopore deviceaccording to one embodiment.

FIGS. 7A-7E illustrate a method for manufacturing a 3D nanopore deviceaccording to another embodiment.

In order to better appreciate how to obtain the above-recited and otheradvantages and objects of various embodiments, a more detaileddescription of embodiments is provided with reference to theaccompanying drawings. It should be noted that the drawings are notdrawn to scale and that elements of similar structures or functions arerepresented by like reference numerals throughout. It will be understoodthat these drawings depict only certain illustrated embodiments and arenot therefore to be considered limiting of scope of embodiments.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS Exemplary NanoporeDevices

As described above, current state-of-art nanopore devices are limited atleast in terms of sensitivity and manufacturing cost. The nanoporedevice embodiments described herein address, inter alia, theselimitations of current nanopore devices.

FIG. 2A-2D schematically depict various views of a nanopore device 200incorporating solid-state nanopore technology with a three dimensional(“3D”) array architecture according to one embodiment. As shown in FIG.2A, the device 200 includes a plurality of 2D arrays or layers 202A-202Estacked along a Z axis 204. While the 2D arrays 202A-202E are referredto as “two dimensional,” each of the 2D arrays 202A-202E has somethickness along the Z axis. FIG. 2B depicts a top view of the top 2Darray 202A depicted in FIG. 2A. FIGS. 2C and 2D schematically depictfront and right side views of the nanopore device 200 depicted in FIG.2A.

The top 2D array 202A includes first and second selecting (inhibitoryelectrode) layers 206, 208 configured to direct movement of chargedparticles (e.g., biopolymers) through the nanopores 210 (pillars) formedin the first and second selecting layers 206, 208. The first selectinglayer 206 is configured to select from a plurality of rows (R1-R3) inthe 2D array 202A. The second selecting layer 208 is configured toselect from a plurality of columns (C1-C3) in the 2D array 202A. In oneembodiment, the first and second selecting layers 206, 208 select fromthe rows and columns, respectively, by modifying a charge adjacent theselected row and column and/or adjacent to the non-selected rows andcolumns. The other 2D arrays 202B-202E include rate control/currentsensing electrodes. Rate control electrodes may be made of highlyconductive metals, such as Au—Cr, TiN, TaN, Pt, Cr, Graphene, Al—Cu,etc. The rate control electrodes may have a thickness of about 2 toabout 1000 nm. Rate control electrodes may also be made in thebiological layer in hybrid nanopores.

Hybrid nanopores include a stable biological/biochemical component withsolid state components to form a semi-synthetic membrane porin toenhance stability of the nanopore. For instance, the biologicalcomponent may be an αHL molecule. The αHL molecule may be inserted intoa SiN based 3D nanopore. The αHL molecule may be induced to take on astructure to ensure alignment of the αHL molecule with the SiN based 3Dnanopore by apply a bias to an electrode (e.g., in the top 2D array202A).

The nanopore device 200 has a 3D vertical pillar stack array structurethat provides a much larger surface area for charge detection than thatof a conventional nanopore device having a planar structure. As acharged particle (e.g., biopolymer) passes through each 2D array202A-202E in the device, its charge can be detected with a detector(e.g., electrode) in some of the 2D arrays 202B-202E. Therefore, the 3Darray structure of the device 200 facilitates higher sensitivity, whichcan compensate for a low signal detector/electrode. Further, the highlyintegrated small form factor 3D structure provides a high densitynanopore array while minimizing manufacturing cost.

In use, the nanopore device 200 is disposed in a middle chamberseparating top and bottom chambers (not shown) such that the top andbottom chambers are fluidly coupled by the nanopore pillars 210. Thetop, middle, and bottom chambers include an electrolyte solution (e.g.,Ag, AgCl₂, etc.) containing the charged particles (e.g., DNA) to bedetected. Different electrolyte solutions can be used for the detectionof different charged particles.

Electrophoretic charged particle translocation can be driven by applyinga bias to electrodes disposed in a top chamber (not shown) adjacent thetop 2D array 202A of the nanopore device 200 and a bottom chamber (notshown) adjacent the bottom 2D array 202E of the nanopore device 200. Insome embodiments, the nanopore device 200 is disposed in a middlechamber (not shown) such that the top and bottom chambers are fluidlyand electrically coupled by the nanopore pillars 210 in the nanoporedevice 200. The top, middle, and bottom chambers may contain theelectrolyte solution.

FIG. 3 schematically depicts a nanopore device 300 according to anotherembodiment. FIG. 3 depicts the top 2D array 302 in a cross-sectional(x-z plane) view showing the 3D nanopore 310 and nano-electrode schemes.Each nanopore 310 is surrounded by nano-electrodes 312, allowing thenanopore 310 channel to operate under an electric bias field conditiongenerated using the nano-electrodes 312. Cross-patterned nanogapnano-electrodes 312CS-312Cn, 312RS-312Rn are disposed in two layers ontop of the nanopore device 300. These nano-electrodes 312CS-312Cn,312RS-312Rn are column and row inhibitory nano-electrodes 312CS-312Cn,312RS-312Rn for the nanopore array, respectively. The cross-patternednano-electrodes 312CS-312Cn, 312RS-312Rn as shown in the top 2D array302 (x-y plane view) may be formed/patterned at the metal lithographysteps. Nano-electrodes 312 in the remaining 2D arrays in the 3D stackmay be formed by plane depositing metals. The nanopore 310 hole pillarsare surrounded by the metal nano-electrodes 312CS-312Cn, 312RS-312Rn,and thus may operate under the full influence of the electrical biasapplied to the multiple stacked nano-electrodes 312.

By applying an inhibitory electrical bias (0V-VCC) to select nanogapnano-electrodes 312CS-312Cn, 312RS-312Rn in the top 2D array 302,biomolecular translocation (e.g., electrophoretic) through one or morenanopores 302 in the top 2D nanopore array 302 can be inhibited tocontrol nanopore array operation according to one embodiment. Theelectrical bias applied to the nano-electrodes 312CS-312Cn, 312RS-312Rncan generate an electric field sufficient to suppress ionictranslocation of charged particles (e.g., nucleic acids) from a topchamber (not shown) to a bottom chamber (not shown) in a directionorthogonal to the nano-electrodes 312CS-312Cn, 312RS-312Rn.Nano-electrode 312 mediated ionic translocation suppression can besubstantially complete or the electrical bias can be modulated to onlyreduce the rate of ionic translocation. In one embodiment, after one ormore nanopores 310 are selected (e.g., for DNA biomoleculestranslocation and sequencing), the electrical biases in a stack of 3Dnanopore nano-electrodes 312 can be modulated to control thebiomolecular translocation speed. In one embodiment, the inhibitoryelectrical bias reduces/stops ionic current flow in the verticaldirection to thereby select and/or deselect various columns and rowsdefined by the nanogap nano-electrodes 312CS-312Cn, 312RS-312Rn . At thesame time, the nano-electrodes 312 can detect current modulationsresulting from passage of charged particles (e.g., DNA biomolecules)through the 3D vertical nanopore 310 pillars. In some embodiments, thenano-electrodes 312 can detect current modulations using a variety ofprinciples, including ion blockade, tunneling, capacitive sensing,piezoelectric, and microwave-sensing.

FIG. 4 is a table 400 illustrating the voltage operation of the nanoporedevice 300 depicted in FIG. 3. As shown in FIG. 4, the nanopore device300 can be operated in both translocation and read (sense) mode bymodulating the voltage applied to various electrodes 312.

FIG. 5 schematically illustrates a single 3D nanopore sensor 520 in ananopore device according to one embodiment. The sensor 520 has a columninhibitory electrode layer 522, a row inhibitory electrode layer 524,and a plurality of rate control/sensing electrode layers 526, 528, 530.These layers are stacked on top of each other and separated by aninsulator layer 532 (e.g., SiO₂) to define a vertical nanopore 510 holepillar. Each layer may have a polysilicon or metal (e.g., Ta, Al, Cr,Au—Cr, Ni, Graphene, etc.) top sub-layer and various other sub-layers(e.g., Al₂O₃, Si₃N₄, n⁺ or p⁺ polysilicon, etc.) The 3D nanopore sensor520 can operate on a variety of principles, including ion blockade,tunneling, capacitive sensing, piezoelectric, and microwave-sensing.Rate control/sensing electrode layers 526, 528, 530 can be activated byapplying a rate control or a sensing bias to the respective electrodelayers 526, 528, 530. The sensing electrode layers 526, 528, 530 candetect a change in an electrical characteristic (e.g., an electrodecurrent and/or a tunneling current).

3D nanopore devices (e.g., 200, 300) allow either direct or targetedsequencing in an array while minimizing form-factor overhead, becausethe 2D arrays 202, 302 in the nanopore devices 200, 300 can be stackedvertically instead of positioned horizontally, thereby allowing for highdensity applications. Further, 3D nanopore devices (e.g., 200, 300) arescalable, with medium to large 3D nanopore devices having more than1,000 nanopore 210, 310 pillars. Consequently, a larger number ofsequencing sensors can be accommodated within the same form-factor. 3Dnanopore devices (e.g., 200, 300) can also incorporate biologicalnanopore or hybrid nanopore technologies to provide more architecturalflexibility to accommodate a user's needs.

In 3D nanopore devices (e.g., 300), each nanopore 310 pillar is composedof a stack of nano-electrodes 312 defining a plurality of nanopores 310.As such, the effective surface area of the sensors in each nanopore 310column can be orders of magnitude greater than the surface area of asingle sensor. In one embodiment, the effective sensor surface area canbe 2-3 orders of magnitude greater than the surface area of a singlesensor. This increase in effective sensor surface area can significantlyimprove the sensor signal to noise ratio and sensitivity, whileminimizing manufacturing costs.

Exemplary Nanopore Device Manufacturing Methods

3D nanopore devices (e.g., 200, 300) can be manufactured utilizing manydifferent methods. In one embodiment, a semiconductor technology (e.g.,CMOS process, described below) is used to manufacture 3D nanoporedevices 200, 300. The CMOS process also allows nanopore 310 width to betunable using a large nanopore array. In one embodiment, nanopore 310width can be controlled during manufacturing using software with a lookup table, allowing for mass production manufacturing. Using a CMOSprocess can embed biosensor solutions in CMOS technology. In variousembodiments, the CMOS process includes a 2-dim well for electrochemicalreactions and/or an ion-sensitive filed effect transistor technology.Microfluidic channels can be integrated into the 3D nanopore devices200, 300 (e.g., within a die), therefore reducing the cost of thedevices 200, 300.

FIGS. 6A-6E illustrate a method 600 of manufacturing a nanopore deviceaccording to one embodiment. As shown in FIG. 6A, a first dielectricbase layer (e.g., SiO₂, Al₂O₃, etc.) 602A, a first base layer of Si₃N₄604A and a first layer of a metal (e.g., Au—Cr, Al, Graphene, etc.) orpolysilicon layer 607A are deposited on top of each other. Then secondand third layers of dielectric base, base, and metal 602B, 604B, 607B,602C, 604C, 607C are deposited on top of each other and the previouslayers. For instance, these deposition steps can be performed usingchemical vapor deposition (“CVD”) and/or Atomic Layer Deposition (“ALD”)of base dielectric layers 602, trimming dielectric layers, and/ormembrane dielectric layers (see FIG. 6C below). The first dielectricbase layer 602A may have a thickness of about 100 nm to 1000 nm toreduce substrate coupled low level noise.

As shown in FIG. 6B, a nanopore 610 is then etched into the depositedlayers (e.g., a using high aspect ratio (above 5) nanopore hole trenchetching process). The high aspect ratio etching can provide a sharpshape to a trench profile of the nanopore 610 channel pillar. Totaldepth of the nanopore pillar can be few hundred nanometers to severalmicrons depending on the applications.

Next, as shown in FIG. 6C, thin layers of a dielectric coating 612(e.g., Si₃N₄, Al₂O₃, SiO₂, etc.) are deposited (e.g., by atomic layerdeposition “ALD”) on the inner surface of the nanopore 610 to determinethe width of the nanopore 610. The dielectric coating 612 may vary inthickness (e.g., from about 10 nm to about 50 nm). By controlling theamount of dielectric coating 612 deposited on the inner surface of thenanopore 610 (e.g., using ALD) target nanopore 610 widths of about 2 nmto about 100 nm can be achieved. Accordingly, the width/diameter of thenanopore/trench 610 can be controlled using ALD of a dielectric coating612 to suit a variety of applications. A top dielectric coating 612 mayhave a thickness of about 5 nm to about 20 nm. Depending on theapplications and required nanopore 610 opening dimensions, variouslithography techniques (e.g., those used in the mass volume production)can be used to etch the nanopore 610 opening. In addition, the depth ofthe nanopore 610 channel can be selected for the required sensitivityand accuracy with ease using the manufacturing methods described herein.

As shown in FIG. 6D, the vertical nanopore channel and the stackedlayers are etched (see “steps” on right side of stacked layers) to formthe horizontal (X axis) and vertical (Y axis) nanopore channels toprovide access for electrodes 614 (e.g., row and column inhibitoryelectrodes) on top and addressing circuits. The base Si₃N₄ 604A (orAl₂O₃) layers are selectively wet etched to provide electrical access toall of the horizontal electrodes. Finally, the remaining space is filledwith metal.

FIG. 6E depicts the manufactured 3D nanopore device in use forsequencing a biopolymer (e.g., DNA).

FIGS. 7A-7E illustrate a method 700 of manufacturing a nanopore deviceaccording to another embodiment. The methods 600, 700 depicted in FIGS.6A-6E and 7A-7E are similar and share many of the same techniques.

As shown in FIG. 7A, multiple layers of dielectric films (such as SiO₂,Al₂O₃, ZnO, HfO₂, etc.; 10 nm-100 nm) low stress nitride film (Si₃N₄)and metal (Ta, Al, Cr, Ti, Au-Cr, Graphene, etc.; few nm to 100's of nm)and inter-metal layer (SiO₂) collectively 702 are stack deposited on aSi or Quartz substrate using CVD (Low Pressure/Plasma Enhanced) orAtomic Layer Deposition (ALD). Next, a bottom chamber opening 704(5×5˜100×100 μm²) is etched by a Deep Reactive Ion Etching (RIE) or KOHwet etching.

As shown in FIG. 7B, a top metal layer 706 is deposited. Next, a topchamber opening 708 is defined using a high aspect ratio nanopore holedeep trench etching process by Reactive Ion Etching. This process cangenerate nanopore trench etch opening diameters from a few nm to about100 nm. Lithography techniques using colloidal mask (e.g., Nano-dots,Quantum-dots, or Graphene oxide pores) can also be used instead ofconventional tools.

As shown in FIGS. 7C and 7D, Thin layers of dielectrics 610 (e.g.,films) also can be deposited by ALD to trim the nanopore width toachieve target nanopore widths (using ALD) from about 2 nm to about 1000nm. Nanopore channel widths can also be controlled to have variabletrench width, allowing variable nanopore width for the target diameter.

FIG. 7E depicts the manufactured 3D nanopore device in use forsequencing a biopolymer (e.g., DNA).

The 3D nanopore device may facilitate multiplex sequencing applicationsusing high density low cost nanopore channels. A number of electrodes inthe 3D nanopore device may be selected depending on the requiredsequencing applications to provide a Time of Flight (“TOF”) technique tocontrol the translocation speed by controlled biasing. Controlling thetranslocation speed may improve reading of DNA molecules and improves asensitivity of the sensor. The 3D nanopore device may also facilitate anelectro-chemical, thermal, or electro-optical reaction to take place inan enlarged, separated nanopore well with a multi-electrode system toenhance electrochemical and/or sequencing reactions. The 3D nanoporedevice may further facilitate multiplex sequencing using a multi-arrayconfiguration wherein individual nanopore channel pillars areaddressable. Moreover, the 3D nanopore device may further facilitatestandard qPCR within a nanopore channel pillar and/or probe-mediatedtargeted sequencing. In addition, the 3D nanopore channel opening widthis tunable for different applications. In one embodiment, the nanoporechannel opening width is tunable from about 1 nm to about 100 nm. Thenanopore channel opening width may be electronically tunable duringmanufacturing. The 3D nanopore devices described herein can be used inthe detection of various charged particles, including but not limited tobiomolecules such as nucleotides, nucleic acids, and proteins (directdetection). The 3D nanopore devices can also be used in DNA sequencingand detection of protein-DNA interactions.

Manufacturing metal or polysilicon plane based nanopore arrays usinglithographic processes (e.g., Through-Silicon Via (“TSV”) fabrication)minimizes manufacturing cost and line resistance (which significantlyreduces IR drop and RC delay limitations to scaling).

The corresponding structures, materials, acts and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structures, materials, acts and equivalents for performingthe function in combination with other claimed elements as specificallyclaimed. It is to be understood that while the invention has beendescribed in conjunction with the above embodiments, the foregoingdescription and claims are not to limit the scope of the invention.Other aspects, advantages and modifications within the scope to theinvention will be apparent to those skilled in the art to which theinvention pertains.

Various exemplary embodiments of the invention are described herein.Reference is made to these examples in a non-limiting sense. They areprovided to illustrate more broadly applicable aspects of the invention.Various changes may be made to the invention described and equivalentsmay be substituted without departing from the true spirit and scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processact(s) or step(s) to the objective(s), spirit or scope of the presentinvention. Further, as will be appreciated by those with skill in theart that each of the individual variations described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinventions. All such modifications are intended to be within the scopeof claims associated with this disclosure.

Any of the devices described for carrying out the subject diagnostic orinterventional procedures may be provided in packaged combination foruse in executing such interventions. These supply “kits” may furtherinclude instructions for use and be packaged in sterile trays orcontainers as commonly employed for such purposes.

The invention includes methods that may be performed using the subjectdevices. The methods may comprise the act of providing such a suitabledevice. Such provision may be performed by the end user. In other words,the “providing” act merely requires the end user obtain, access,approach, position, set-up, activate, power-up or otherwise act toprovide the requisite device in the subject method. Methods recitedherein may be carried out in any order of the recited events which islogically possible, as well as in the recited order of events.

Exemplary aspects of the invention, together with details regardingmaterial selection and manufacture have been set forth above. Otherdetails of the present invention, these may be appreciated in connectionwith the above-referenced patents and publications as well as generallyknown or appreciated by those with skill in the art. The same may holdtrue with respect to method-based aspects of the invention in terms ofadditional acts as commonly or logically employed.

In addition, though the invention has been described in reference toseveral examples optionally incorporating various features, theinvention is not to be limited to that which is described or indicatedas contemplated with respect to each variation of the invention. Variouschanges may be made to the invention described and equivalents (whetherrecited herein or not included for the sake of some brevity) may besubstituted without departing from the true spirit and scope of theinvention. In addition, where a range of values is provided, it isunderstood that every intervening value, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention.

Also, it is contemplated that any optional feature of the inventivevariations described may be set forth and claimed independently, or incombination with any one or more of the features described herein.Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin claims associated hereto, the singular forms “a,” “an,” “said,” and“the” include plural referents unless the specifically stated otherwise.In other words, use of the articles allow for “at least one” of thesubject item in the description above as well as claims associated withthis disclosure. It is further noted that such claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Without the use of such exclusive terminology, the term “comprising” inclaims associated with this disclosure shall allow for the inclusion ofany additional element—irrespective of whether a given number ofelements are enumerated in such claims, or the addition of a featurecould be regarded as transforming the nature of an element set forth insuch claims. Except as specifically defined herein, all technical andscientific terms used herein are to be given as broad a commonlyunderstood meaning as possible while maintaining claim validity.

The breadth of the present invention is not to be limited to theexamples provided and/or the subject specification, but rather only bythe scope of claim language associated with this disclosure.

1. A 3D nanopore device for characterizing biopolymer molecules,comprising: a first Si₃N₄ layer deposited on a first dielectric layer; afirst metal layer deposited on the first Si₃N₄ layer; a seconddielectric layer deposited on the first metal layer; a second Si₃N₄layer deposited on the second dielectric layer; a second metal layerdeposited on the second Si₃N₄ layer; a third dielectric layer depositedon the second metal layer; a third Si₃N₄ layer deposited on the thirddielectric layer; a third metal layer deposited on the third Si₃N₄layer; a first plurality of elongate gate electrodes etched andpatterned into the second metal layer; and second plurality of elongategate electrodes etched and patterned into the third metal layer, whereineach of the first plurality of elongate gate electrodes is disposed inthe second metal layer, wherein each of the first plurality of elongategate electrodes is parallel to the other elongate gate electrodes of thefirst plurality of elongate gate electrodes, wherein each of the secondplurality of elongate gate electrodes is parallel to the other elongategate electrodes of the second plurality of elongate gate electrodes, andwherein the first and second pluralities of elongate gate electrodes areorthogonal to each other along the second and third metal layersrespectively.
 2. The system of claim 1, further comprising a pluralityof nanopore channels etched into the first and second pluralities ofparallel elongate gate electrodes, the second and third Si₃N₄ layers,and the second and third dielectric layers.
 3. The system of claim 2,further comprising a bottom chamber fluidly coupled to the plurality ofnanopore channels.
 4. The system of claim 2, further comprising a middlechamber between top and bottom chambers, wherein the first dielectriclayer, the first Si₃N₄ layer, the first metal layer, the seconddielectric layer, the second Si₃N₄ layer, and the second metal layer aredisposed in the middle chamber; and an electrolyte solution disposed inthe top, middle, and bottom chambers, such that the top and bottomchambers are fluidly coupled by the plurality of nanopore channels. 5.The system of claim 4, further comprising top and bottom chamberelectrodes disposed in the top and bottom chambers respectively, whereinthe top and bottom chamber electrodes each comprise Ag/AgCl₂.
 6. Thedevice of claim 4, wherein the electrolyte solution comprises KCl orLiCl₂.
 7. The system of claim 2, further comprising top and innersurface dielectric coatings deposited on an inner surface of a nanoporechannel of the plurality of nanopore channels to functionalize the innersurface of the nanopore channel of the plurality of nanopore channelsfor a biomolecular interaction.
 8. The system of claim 7, wherein thetop and inner surface dielectric coatings form a gate electrodedielectric for sensing.
 9. The system of claim 7, wherein the top andinner surface dielectric coatings are configured to adjust a width ofthe nanopore.
 10. The system of claim 7, wherein the inner dielectriccoating comprises Al₂O₃, SiO₂, ZnO, or HfO.
 11. The system of claim 7,wherein the inner dielectric coating has a thickness of about 10 nm toabout 50 nm.
 12. The system of claim 7, wherein the top dielectriccoating comprises Si₃N₄, Al₂O₃, or SiO₂.
 13. The system of claim 7,wherein the top dielectric coating has a thickness of about 5 nm toabout 50 nm.
 14. The system of claim 2, wherein each of the plurality ofnanopore channels has respective diameters from about 2 nm to about 100nm.
 15. The system of claim 1, further comprising a stack of dielectriclayers, including the second and third dielectric layers; a stack ofSi₃N₄ layers, including the second and third Si₃N₄ layers; a stack ofmetal layers, including the second and third metal layers; and ananopore channel etched into the respective stacks of dielectric layers,Si₃N₄ layers, and metal layers.
 16. The system of claim 1, wherein thefirst plurality of elongate gate electrodes are a first plurality ofinhibitory electrodes.
 17. The system of claim 16, wherein the secondplurality of elongate gate electrodes are a second plurality ofinhibitory electrodes.
 18. The system of claim 17, wherein the first andsecond pluralities of inhibitory electrodes form an array partiallydefining the plurality of a plurality of nanopore channels.
 19. Thesystem of claim 1, further comprising a sensing electrode layer.
 20. Thesystem of claim 19, wherein the sensing electrode layer is configured tooperate by ion blockade, tunneling, capacitive sensing, piezoelectric,or microwave-sensing.